Gene therapy methods are currently being developed that safely and persistently deliver therapeutically effective quantities of gene products to patients. Using these methods, a nucleic acid molecule can be introduced directly into a patient (in vivo gene therapy), or into cells isolated from a patient or a donor, which are then subsequently returned to the patient (ex vivo gene therapy). The introduced nucleic acid then directs the patient's own cells or grafted cells to produce the desired therapeutic product. Gene therapy also allows clinicians to select specific organs or cellular targets (e.g., muscle, blood cells, brain cells, etc.) for therapy.
Nucleic acids may be introduced into a patient's cells in several ways, including viral-mediated gene delivery, naked DNA delivery, and transfection methods. Viral-mediated gene delivery has been used in a majority of gene therapy trials. C. P. Hodgson Biotechnology (1995) 13:222-225. The recombinant viruses most commonly used are based on retrovirus, adenovirus, herpesvirus, pox virus, and adeno-associated virus (AAV).
Recombinant adeno-associated viral vectors hold promise as gene delivery vectors for human gene therapy. However, one significant obstacle to using such vectors as drugs is the development of a truly scaleable process to produce and purify the vector at commercially viable levels. For a review of the challenges involved in scaling AAV vector production for commercial use, see Qu and Wright, Cur. Opin. Drug Disc. and Develop. (2000) 3:750-755. Recently, several potentially scalable column chromatography techniques to purify rAAV virions have been developed. While these column chromatography-based purification methods have demonstrated that rAAV virions can be purified at large scale, the preparation of purified virions using column chromatography contains a significant amount of AAV empty capsids. The typical ratio of empty capsids to virions containing a heterologus gene of interest (“AAV vector particles”) is about 10 or higher, i.e., approximately 90% of the recovered vectors are empty capsids.
The presence of a large amount of empty capsids may hinder clinical applications, e.g., by eliciting unwanted immune responses to the capsid protein or by competing for target cell surface binding sites. Consequently, techniques have been developed to remove the empty capsids from rAAV virion preparations. These techniques typically rely on ultracentrifugation, for example gradient centrifugation in cesium chloride or iodixanol. Such centrifugation techniques are labor intensive, typically result in low vector yield, and are not scalable. Kaludov et al., (2002) Hum. Gene Ther. 13:1235-1243, describe methods of purifying rAAV-2, -4 and -5 vectors using anion exchange columns. However, the experimenters were only able to recover 2%, 0.6% and 6.3%, respectively, as packaged genomes, even after pooling the eluates and concentrating the fractions.
Thus, there remains a need for new ways of eliminating or reducing the numbers of empty capsids from stocks of AAV vector particles so that manufacturing capability is enhanced.